This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Mitotic spindles can show remarkable variability between tissues and organisms, but there is currently little understanding of the evolutionary basis of this diversity. Thus it is unclear why metaphase spindles in different Eukaryotes exhibit a range of morphologies and the volumes of these spindles vary over one thousand fold. Here, we propose a comparative study to investigate the evolutionary forces that shape the mitotic spindle. We will use one-cell nematode embryos in mitosis to address three broad questions: 1) What is the extent of variation in spindle dynamics and structure? We will investigate both small and large phylogenic distances by studying ~60 species from across the nematode phylum - including all known species of the Caenorhabditis genus. This work will use a number of light microscopy techniques to determine the range of spindle variation in nematodes. 2) What is the biophysical basis of this variation? We will perform an in-depth analysis of microtubule dynamics and spindle architecture in ~10 select species. Fluorescence techniques (when possible in nonmodel species), thin-section and 3-D electron microscopy, and perturbations experiments, both physical and genetic (when feasible), will be used to measure the dynamics, number and behavior of microtubules. We will develop biophysical models to explain how differences in microtubule behavior lead to differences in spindle architecture. 3) What is the evolutionary basis of this variation? We will use models of phenotypic evolution to gain insight into: (i) the extent of phylogenetic effects;(ii) the dependence of spindle features on cell size, karyotype, life history, and other attributes;and (iii) the importance of selection and drift in shaping various components of the spindle. A parallel study on one species, Caenorhabditis elegans, will provide a more detailed analysis of evolutionary forces: we will use mutation accumulation lines to measure how spontaneous mutations modify the mitotic spindle, and we will determine how this variability is shaped over evolution by comparing these lines with ~40 different wild isolates. This unique comparative approach combines molecular methods, quantitative microscopy, and mathematical modeling, to develop a comprehensive model of how the mitotic spindle is shaped through evolution by mutation, selection, drift, and cellular biophysics.